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Tropical filamentous marine cyanobacteria have emerged as a viable source of novel bioactive natural products for drug discovery and development. In the present study, aplysiatoxin (1), debromoaplysiatoxin (2) and anhydrodebromoaplysiatoxin (3), as well as two new analogues, 3-methoxyaplysiatoxin (4) and 3-methoxydebromoaplysiatoxin (5), are reported for the first time from the marine cyanobacterium Trichodesmium erythraeum. The identification of the bloom-forming cyanobacterial strain was confirmed based on phylogenetic analysis of its 16S rRNA sequences. Structural determination of the new analogues was achieved by extensive NMR spectroscopic analysis and comparison with NMR spectral data of known compounds. In addition, the antiviral activities of these marine toxins were assessed using Chikungunya virus (CHIKV)-infected cells. Post-treatment experiments using the debrominated analogues, namely compounds 2, 3 and 5, displayed dose-dependent inhibition of CHIKV when tested at concentrations ranging from 0.1 µM to 10.0 µM. Furthermore, debromoaplysiatoxin (2) and 3-methoxydebromoaplysiatoxin (5) exhibited significant anti-CHIKV activities with EC50 values of 1.3 μM and 2.7 μM, respectively, and selectivity indices of 10.9 and 9.2, respectively.

Prokaryotic filamentous marine cyanobacteria have emerged as an important source of structurally novel natural products in drug discovery efforts [1,2,3]. To date, more than 450 compounds have been reported with a majority belonging to either the non-ribosomal peptide synthetase (NRPS) or hybrid polyketide synthase (PKS)-NRPS class [1,2,3]. A number of these compounds possess potent biological activities with specific drug targets, including Class 1 histone deacetylase (e.g., largazole), proteasome (e.g., carmaphycins A and B), voltage-gated sodium channels (e.g., kalkitoxin and hoiamide A), and falcipains (e.g., symplostatin 4). Due to their specific interactions with these molecular targets, marine cyanobacterial compounds are an attractive source of lead compounds for drug development, particularly in the area of cancer, neurological disorders, and infectious diseases [4].

Aplysiatoxin and related analogues, e.g., oscillatoxins and nhatrangins, are distinct polyketide classes of marine toxins isolated from several cyanobacterial species, including Lyngbya majuscula, Schizothrix calcicola, and Oscillatoria nigro-viridis [5,6,7]. Several aplysiatoxin-related analogues are known to possess potent tumor-promoting properties through the activation of protein kinase C (PKC) [8]. PKC belongs to a family of serine/threonine kinases and plays important roles in cell proliferation, differentiation and apoptosis [9]. Due to its importance in cellular signal transduction pathways, PKC is a potential drug target for the treatment of various diseases, particularly in cancer therapy [10]. Researches from Japan recently developed new simplified analogues based on aplysiatoxin class of molecules, e.g., aplog-1, with potent anti-proliferative properties as potential anticancer agents [11,12,13,14,15,16,17,18,19]. These synthetic analogues have been shown to inhibit the action of tumor promotors as well as prevent growth of cancer cells in similar ways to bryostatin 1 [11].

Chikungunya virus (CHIKV) is a mosquito-transmitted Alphavirus that has re-emerged as a significant public health threat in recent years [20]. CHIKV is the causative agent of chikungunya fever, which is characterized by myalgia, arthralgia, headaches, nausea, fever, and maculopapular rash [21]. In addition, recurrent myalgia and arthralgia have been known to persist for years after the infection clears in some patients [22,23]. The widening geographical distribution of the mosquito vectors, Aedes albopictus and Aedes aegypti has also led to researchers suggesting the possibility of CHIKV being established almost globally [24]. Despite this, there is currently no antiviral treatment or vaccine against CHIKV [25].

Three known aplysiatoxins (1–3), as well as new analogues, compounds 4 and 5, were isolated from the bioactive organic extracts of Trichodesmium erythraeum (TLT/PSK/001), recently collected from Pulau Seringat Kias, Singapore (Figure 1). Herein, we describe the structure elucidations of two new aplysiatoxin analogues, 3-methoxyaplysiatoxin (4) and 3-methoxydebromoaplysiatoxin (5), and report anti-CHIKV activities for compounds 2, 3, and 5.

Figure 1

Aplysiatoxin-related compounds from Trichodesmium erythraeum.

2. Results and Discussion

Phylogenetic analysis based on the 16S rRNA gene sequences identified the marine cyanobacterium as Trichodesmium erythraeum. Subsequent chemical investigation on the organic extract of Trichodesmium erythraeum (TLT/PSK/001) resulted in the isolation of known compounds, aplysiatoxin (1), debromoaplysiatoxin (2) and anhydrodebromoaplysiatoxin (3), as well as two new analogues, 3-methoxyaplysiatoxin (4) and 3-methoxydebromoaplysiatoxin (5) (Figure 1). The new aplysiatoxin analogues were isolated as colorless amorphous powder.

Extensive 1D and 2D NMR spectroscopic analysis of compounds 4 and 5 as well as comparison with NMR spectral data of known compounds revealed structural similarities to aplysiatoxin (1) and debromoaplysiatoxin (2), respectively. The presence of an additional methoxy group in either compound 4 or 5 was observed in the 1H and 13C NMR spectra, namely δ 3.27/3.27 (H-27) and δ 49.40/49.28 (C-27), respectively. In addition, the placement of the methoxy group at C-3 was revealed by a distinct correlation from H-27 to C-3 in the HMBC spectra of compounds 4 and 5. Due to the structural similarities with aplysiatoxin and debromoaplysiatoxin, the absolute stereochemistry of the new methoxy analogues is assumed to be identical. The new methoxy analogues of aplysiatoxin and debromoaplysiatoxin are likely to be natural products and not artefacts. This is due to the relative amounts of 4 and 5 compared with 1 and 2, respectively. In addition, it has been reported that aplysiatoxin is chemically unstable due to the labile hemiacetal hydroxyl group at C-3 and is easily eliminated to form compound 3 under weak acidic conditions [6]. Under such acidic conditions, it is unlikely that the tertiary carbocation formed at C-3 is attacked by solvent MeOH to form the methoxy functionality. Furthermore, steric hindrance by neighboring carbons could prevent the entry of MeOH to form a methoxy group at C-3.

The anti-CHIKV activities of compounds 1 to 5 were evaluated in both pre- and post-treatment studies. Pre-treatment studies were performed to determine the effects of the compounds on CHIKV binding and entry processes. In pre-treatment studies, BHK21 cells were treated with the compounds before being infected with CHIKV. Cell culture supernatants were harvested at 24 hpi (hours post-infection) for determination of viral titres via plaque assays. The effects of the marine-derived compounds on CHIKV replication within infected cells were assessed in post-treatment studies. For post-treatment studies, CHIKV-infected cells were treated with the compounds before cell culture supernatants were collected for virus titration at 24 hpi. Three compounds, 2, 3 and 5, displayed significant dose-dependent inhibition of CHIKV titres upon post-treatment (Figure 2), while compounds 1 and 4 did not result in any significant inhibition at 10 μM. Post-treatment resulted in statistically significant reduction in infectious CHIKV titre at treatment concentrations of 1 μM onwards for compounds 2 and 5, and a concentration of 10 μM for compound 3. Compound 2 displayed the most potent antiviral activity upon post-treatment with an EC50 value of 1.3 μM and a selectivity index (SI = CC50/EC50) of 10.9 (Table 2). The EC50 values of compounds 3 and 5 were 22.3 μM (SI of 11.4) and 2.7 μM (SI of 9.2), respectively (Table 2).

Pre-treatment of cells with compounds 1 to 5 prior to CHIKV infection did not result in any significant inhibition, except at a high concentration of 10 μM for compounds 2 and 5 (Figure 2). The minimal inhibition observed may have been due to the high treatment concentrations, which allowed the compounds to persist at sufficient levels in the cells to cause inhibition even after the pre-treatment period. Taken together, results from the pre-treatment and post-treatment studies suggested that the antiviral mechanism of compounds 2, 3 and 5 is likely to target a step in the CHIKV replication cycle that occurs after viral entry. To ensure that the decreases in infectious titres were not due to cell cytotoxicity upon compound treatment, cell viability assays were performed using alamarBlue® cell viability assay. Cell viability remained above 70% for all concentrations of the compounds assayed indicating minimal cytotoxicity (Figure 2).

This study represents the first report of antiviral activities of the aplysiatoxin-class of molecules. Debromoaplysiatoxin (2) and 3-methoxydebromoaplysiatoxin (5) were particularly active. Based on the activity profile, it is interesting to note that the absence of the bromine atom in 2 and 5 enhanced their antiviral properties. In addition, these compounds displayed anti-CHIKV effects at concentrations that resulted in minimal cytotoxicity. Debromoaplysiatoxin (2) and 3-methoxydebromoaplysiatoxin (5) may therefore represent a novel class of antivirals and further investigations into essential pharmacophores may allow generation of modified leads with improved pharmacological properties. Further studies in suitable cellular and murine models are warranted to determine their antiviral mechanism, as well as potential as therapeutics against CHIKV.

3. Experimental Section3.1. General Experimental Procedures

A NIKON Eclipse TS100/TS100F inverted microscope was used for morphological characterization of the marine cyanobacterial samples. Proton, 13C, and 2D NMR spectra were recorded in CDCl3 on a 400 MHz Bruker NMR spectrometer using the residual solvent signals (δH at 7.26 ppm and δC at 77.36 ppm) as internal standards. Chemical shifts are reported in ppm and coupling constants (J) are reported in Hz. HRESIMS data were obtained using a Bruker Daltonics MicrOTOF™ series 89 mass spectrometer equipped with an ESI multimode ion source detector. HPLC isolation and purification of compounds 1 to 5 were conducted on a Shimadzu LC-8A preparative LC coupled to a Shimadzu SPD-M10A VP diode array detector. Optical rotations and UV spectra were measured on a Bellingham Stanley ADP 440 polarimeter and a Varian Cary 50 UV visible spectrophotometer, respectively.

3.2. Marine Cyanobacterial Collection

About 1.0 L of the benthic filamentous marine cyanobacterium, Trichodesmium erythraeum, were collected from the C-shaped manmade lagoon (N 1°13′43.56′′, E 103°51′14.69′′) at Pulau Seringat Kias, Singapore, during low tides on 7 August 2012. Small amounts of cyanobacterial samples were rinsed off with distilled water before examination under microscope (10× magnification) for morphological characterization. Remaining samples were stored in 70% aqueous ethanol at −20 °C, before chemical workup. A voucher specimen of this microalga is maintained at NIE under the code TLT/PSK/001. Additional cyanobacterial samples were preserved in 10 mL Nalgene tubes containing RNAlater® solution (Life Technologies Corporation, Carlsbad, CA, USA) for subsequent DNA extraction.

All purified PCR products of 27F and 809F primers were modified and sequenced by using the M13 forward primer 5′-TGTAAAACGACGGCCAGT-3′ and M13 reverse primer 5′-CAGGAAACAGCTATGACC-3′ (1st BASE, Singapore) and tailing the 5′ end in order to allow direct sequencing of 16S rRNA gene by the BigDye® Terminator v3.1 Cycle Sequencing Kit and an ABI 3730 DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA). Sequencing reactions and cleanup were performed according to 1st BASE instructions. Sequences were aligned using online analysis tools, SEQ MATCH, from Ribosomal Database Project (RDP) with the following set of parameters strain (Both), Source (Isolates), Size (≥1200) and Quality (Good). Phylogeny analysis was carried out using the TREE BUILDER tool.

The cytotoxicity profiles of compounds 1 to 5 were assessed by alamarBlue cell viability assay (Invitrogen). In brief, SJCRH30 were seeded in 96-well plates at a density of 1.5 × 104 cells per well and incubated overnight at 37 °C with 5% CO2. Compounds were diluted in RPMI with 2% FCS prior to being incubated with SJCRH30 cell monolayers for 24 h at 37 °C with 5% CO2. After the incubation period, the compounds were removed and cells were washed once with PBS (phosphate buffered saline) before being incubated with alamarBlue® reagent diluted in RPMI with 2% FCS at a dilution ratio of 1:10. Fluorescence was detected at an excitation wavelength of 570 nm and an emission wavelength of 585 nm with the Infinite 200 Pro multiplate reader (Tecan, Männedorf, Switzerland). Measurements from cells treated with compounds or 0.1% DMSO solvent (control) were normalized against those from untreated cells. The 50% cytotoxic concentration (CC50) was determined as the treatment concentration that resulted in 50% cell viability.

3.6.3. Dose-Dependent Anti-Chikungunya Assay

The anti-CHIKV activities of compounds 1 to 5 were investigated using dose-dependent drug treatments on SJCRH30 cells. SJCRH30 cells were seeded at a density of 1.5 × 104 cells per well in 96-well plates and incubated overnight at 37 °C with 5% CO2. For both pre- and post-treatment assays, each compound was diluted in RPMI with 2% FCS. For pre-treatment assays, cells were treated with each compound for 2.5 h at 37 °C prior to being washed once with PBS. Treated cells were incubated with CHIKV-122508 at an MOI (multiplicity of infection) of 10 for 1.5 h to allow viral attachments to the cells. After the adsorption period, cell monolayers were washed twice with PBS before being incubated in RPMI with 2% FCS at 37 °C and 5% CO2. After 24 h, cell culture supernatants were harvested for titration using plaque assay. For post-treatment assays, SJCRH30 cells were infected with CHIKV-122508 at an MOI of 10 for 1.5 h prior to being washed twice with PBS. Infected monolayers were then incubated with varying concentrations of each compound for 24 h at 37 °C with 5% CO2 before supernatants were collected for plaque assay. The 50% effective concentration (EC50) was determined as the concentration that produced 50% of maximal inhibition of log10 virus titre when compared against the solvent control.

3.6.4. Plaque Assay

For quantification of infectious virus titer, BHK21 cells were seeded onto 24-well plates and incubated overnight at 37 °C with 5% CO2. Supernatants from pre- and post-treatment assays were diluted in 10-fold dilutions in RPMI with 2% FCS before being used to infect the cells for 1.5 h at 37 °C. Infected monolayers were washed twice with PBS and overlaid with 1% carboxymethyl cellulose (CMC) in RPMI with 2% FCS. Plaque assay plates were incubated for 3 days at 37 °C with 5% CO2. After the incubation period, CMC was removed and cells were stained and fixed with 10% paraformaldehyde–1% crystal violet (Sigma-Aldrich Chemical, St. Louis, MO, USA) solution for visualization and counting of plaques. Virus titers were expressed as plaque-forming units (PFU) per milliliter.

3.6.5. Statistical Analysis

Data from dose-dependent studies were evaluated for significance by one-way analysis of variance (ANOVA) tests. For samples that showed statistical significance (p < 0.05), a Dunnett’s post-test was performed to compare data from compound treatment against the solvent (0.1% DMSO) control in order to determine the compound concentrations which resulted in significant inhibition of viral titre.

4. Conclusions

Aplysiatoxin-related compounds, including two new analogues, 3-methoxyaplysiatoxin (4) and 3-methoxydebromoaplysiatoxin (5), have been reported for the first time from the marine cyanobacterium, Trichodesmium erythraeum. These compounds, particularly the debromo analogues 2 and 5, showed significant anti-CHIVK activities in post-treatment of infected SJCRH30 cells with EC50 values of 1.3 and 2.7 μM, respectively. The mode of action of the antiviral compounds is likely to target a step in the CHIKV replication cycle that occurs after viral entry. Aplysiatoxins may therefore represent a novel class of antiviral agents and warrant further biological investigation into their mode of action.

Acknowledgments

The authors would like to acknowledge the NIE AcRF (RI 11/10 TLT) and MINDEF DIRP-DSTA grant (R182-000-210-232) for funding this study. In addition, the authors would like to acknowledge Maitreyee Bagchi for help in collection of cyanobacterial samples, Pat Gread (University of Waikato, New Zealand), Angela Ah Buan Lim-Ang, and Anna Say Guek Yee-Lee (NSSE, NIE) for providing valuable technical assistance.

Conflicts of Interest

The authors declare no conflict of interest.

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